1. Field of the Invention
This invention relates to the field of disease diagnosis and also to the fields of genetic engineering and antibody production. More particularly it relates to the identification of virulent and non-virulent strains of Toxoplasma gondii by antibody binding and restriction fragment length polymorphism and to the preparation of compositions, particularly monoclonal antibodies, useful for such identification.
2. Description of the Background
Toxoplasmosis is caused by the protozoan parasite, Toxoplasma gondii. In humans, the disease is traditionally associated with the developing fetus in whom it can cause severe neurological problems manifesting as hydrocephaly, mental retardation and/or blindness [1, 2]. In healthy adults, the disease is typically mild, producing few if any symptoms. In immunocompromised adults, however, the parasite can cause severe or even fatal disease [3, 4, 5]. The disease also occurs in other mammals and is the leading cause of spontaneous abortion in sheep.
The parasite itself is extremely widespread and is typically acquired through the ingestion of undercooked meat in which tissue cysts containing the parasite may reside. Alternatively, the parasite can be contacted through ingestion of cat feces which contain the product of the complete sexual cycle, the oocyst. Both cyst forms are stable (the oocysts particularly so) and so avoidance of infection is difficult. In the U.S.A., serological studies indicate that about 15% of the population has had contact with the parasite [6]. In countries where eating lightly cooked meat is more common, this figure can rise to as much as 85% (e.g., in France [7]). The incidence of disease in the developing fetus is, fortunately, not as high as these figures might at first suggest because it appears that the fetuses of women who contract and control the disease prior to pregnancy are generally not at risk [8].
Diagnosis of congenital infection has in the past relied on serology (reviewed in [14]). This can be done postnatally or, ideally, pre-natally and relies on the relative titers of IgG and IgM (to deduce whether the titers are due to a current infection or legacy of a past infection). The factors contributing to the severity of disease in the developing fetus have been poorly understood. The only well-established factor is that the time of initial infection of the mother relative to conception is critical: infection significantly before conception such that an effective immune response has been mounted by the mother, results in little if any disease. Infection immediately before or after conception (i.e., in the first trimester of pregnancy) results in severe disease for the fetus of about 10-15% of fetuses. Infection late in pregnancy results in relatively less pathology, but occurs in a higher proportion of fetuses. It is unclear what determines whether a given fetus will or will not become diseased. The virulence of the parasite must, by definition, be a factor. Virulence is known to be bimodal, with all known strains of the parasite falling into two classes of relatively benign and highly virulent organisms. However, no simple assay for virulence was known. The direct measurement of virulence has been extremely difficult (requiring inoculation of mice with suspected samples and monitoring the mice for the appearance of the disease) and has not been implemented as part of the diagnostic armamentarium.
In the past two decades, toxoplasmosis has dramatically increased in a relatively new group of patients who are in some way immunodeficient as a result of post-transplantation therapy [5, 9, 10], neoplastic disease [11, 12, 13] or, most recently, acquired immunodeficiency syndrome (AIDS) [3, 4, 5]. In such immunodeficient patients, the parasite can cause a disseminated, potentially fatal form of the disease [5].
AIDS patients with toxoplasmosis typically first present with significant neuropathy (reviewed in [14]). This is due to the fact that one of the tissues most affected by the parasite is the brain wherein massive parasite cysts can be found. Infection is not limited to the brain, however, and tissue cysts can be found throughout the body [11]. The typical routine for diagnosis includes serology, computed tomography, magnetic resonance imaging and/or brain biopsy [1, 15, 16]. Of these, the only definitive route to diagnosis is the brain biopsy as this enables the direct visualization of the parasite, using immuno-peroxidase staining [17].
The course of treatment for toxoplasmosis in pregnant individuals is determined by the stage in pregnancy and whether the infection is acute or chronic. If infection is acute, spiromycin may be administered but is of unproven efficacy. More effective drugs such as combined therapy with pyrimethamine and sulfadiazine are not generally used because of their toxicity for the developing fetus. Hence, these latter drugs are employed in only rare cases where infection of the fetus (as opposed to the mother) is directly demonstrated. Such diagnosis has only been done on an experimental basis.
Treatment of toxoplasmosis in non-pregnant individuals is initiated and maintained with a drug regimen involving a combination of the folate antagonists, pyrimethamine and sulfadiazine [1, 14]. If the disease is identified soon enough, treatment is reasonably effective in combatting the acute disease. However, due to poor tolerance of the drugs, especially of the sulfa compounds in AIDS patients, maintenance on the drug therapy is frequently not possible and recrudescence of the infection is often observed (that is, the drug therapy reduces but does not eliminate the parasite infection).
Accordingly, there remains a need for the development of diagnostic assays that reliably detect and distinguish virulent toxoplasma infection from avirulent toxoplasma infection so that proper selection of a treatment regimen can occur.